Project Summary The goal of this proposal is to investigate mechanisms by which the conducting airways are patterned across the dorsal ventral axis given rise to cartilage and trachealis muscle necessary for appropriate and efficient ventilation movements. While the incidence of airway malacias (airways lacking appropriate cartilage) is high, the etiology of these conditions remains largely unknown. We generated a mouse model of tracheobronchomalacia wherein Wls, a cargo receptor mediating Wnt ligand secretion, was deleted in the epithelium of the trachea. To define mechanisms by which epithelial Wnt signaling promotes tracheal cartilage formation we performed an unbiased analysis of gene expression in prechondrogenic tracheal tissue of control and Wlsf/f;ShhCre embryos. We identified genes that have never been described in respiratory tract, including Lgi3 and Notum, presenting a distinct dorsal-ventral pattern of expression. In Wlsf/f;ShhCre tracheal tissue, genes promoting chondrogenesis, including Bmp ligands, were down regulated, while genes mediating smooth muscle differentiation were upregulated. This proposal will test the hypothesis that Wnt ligands produced by the pulmonary epithelium induce expression of genes promoting chondrogenesis and, simultaneously, induce genes that repress smooth muscle differentiation in the ventral tracheal mesenchyme. The specific aims of this proposal are: 1) To test the hypothesis that a differential spatial gene expression pattern mediated by epithelial Wnt ligands is required for ventral differentiation of tracheal cartilage. This aim will define the spatial expression pattern of novel Wnt target genes in the tracheal tissue and whether Wnt signaling provides positional information in ex vivo assays. Using in vitro assays, this aim will also determine whether novel target genes promote Sox9 and chondrogenesis. 2) To test the hypothesis that genes promoting tracheal smooth muscle development are repressed by epithelial Wnt signaling in the ventral aspect of the trachea. In this aim we will determine via in vivo and in vitro studies whether genes promoting smooth muscle development and upregulated after epithelial deletion of Wls, including Myocd, MyH11, Acta2, and Myo5c, are modulated by Wnt signaling. It will also test whether target genes downregulated after epithelial deletion of Wls are capable to modulate expression of genes promoting smooth muscle. This proposal is innovative, as it will define the role of novel genes expressed in conducting airways in promoting tracheal cartilage or muscle differentiation. In the long term this knowledge will facilitate the development of novel cell based therapies to efficiently treat conditions affecting the normal tracheal patterning and to repair damaged or malformed airways. This proposal will also allow the applicant, a K01 career development awardee, to develop novel hypothesis about patterning of respiratory tract that will be the basis of future R01 proposal. Thus, this project will promote the scientific growth and independence of the applicant.